Jacqueline Ho Division of Pediatric Nephrology Children’s Hospital of Pittsburgh Oct. 9, 2014 SMALL RNAS AND SMALL KIDNEYS

MIRNAS: A NOVEL REGULATORY MECHANISM FOR GENE EXPRESSION ~ miRNAs in humans Up to ½ of all mRNA transcripts are miRNA targets Regulate a variety of processes: Differentiation Proliferation Apoptosis Cell signaling Stem cells and miRNAs

MIRNA BIOGENESIS Ho J and Kreidberg JA (2012); JASN 23(3):

MIRNAS AND ACUTE KIDNEY INJURY Analysis of differential miRNA expression profiles in acute kidney injury: Mouse models: cisplatin, ischemia-reperfusion injury (Lee et al (2014), Kidney Int, epub ahead of print doi: /ki ); (Bellinger et al (2014), PLoSOne, Apr 2; 9(4): e93297) Patients with AKI (Ramachandran et al (2013), Clin Chem, 59(12): ) Possible biomarkers? MicroRNAs as drug targets or drugs themselves Eg. miR-24 antagonism was protective against IRI (Lorenzen et al (2014), JASN, epub ahead of print, pii: ASN ). Eg. miR-126 (overexpression was protective post IRI) (Bijkerk et al (2013), JASN, epub ehead of print, doi: /ASN ).

MIR-17~92 CLUSTER

MIR-17~92 CLUSTER (ONCOMIR-1) Genomic amplification and elevated expression in human B-cell lymphomas Increased expression of miR-17 and miR-106a following IRI (Kaucsar et al (2013), Nucleic Acid Ther; 23(5): ) Deletion of miR-17~92 results in several developmental defects: Lung hypoplasia, ventricular septal defect, impaired B-cell development (Ventura et al (2008), Cell, 132: ) DePontual et al (2011). Nature Genetics, 43(10):

NEPHRON PROGENITORS IN KIDNEY DEVELOPMENT S SP D2D1 Self-renewal: Maintenance of Stem cell pool Multipotent: Generation of differentiated progeny Nephron progenitor

HYPOTHESIS miR-17~92 was expressed in the developing kidney in nephron progenitors Linked to a human syndrome associated with renal anomalies miR-17~92 is required for normal kidney development and function Nephron number? Nephron pattern? Impact on kidney function?

ABLATION OF MIR-17~92 IN NEPHRON PROGENITORS Six2-Cre Mouse miR-17~92 Floxed mouse miR-17~92 miR-17~92 is removedmIR-17~92 function is untouched Six2

LOSS OF MIR-17~92 IN NEPHRON PROGENITORS RESULTS IN RENAL HYPOPLASIA AT P0 ControlHetMutant Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

PRESERVED EXPRESSION OF NEPHRON PROGENITOR MARKERS AT PO Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

FEWER DEVELOPING NEPHRONS (RENAL VESICLES) AT P0 Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

FEWER DEVELOPING GLOMERULI AT P0 Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

NO INCREASE IN APOPTOSIS IN NEPHRON PROGENITORS Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

DECREASED PROLIFERATION OF PROGENITORS Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

MIR-17~92 LOSS IN NEPHRON PROGENITORS Developmental defects: (1) Fewer developing nephrons formed (2) Heterozygous renal hypoplasia (3) Decreased proliferation in nephron progenitors

LONG-TERM CONSEQUENCES : ALBUMINURIA AT 6 WEEKS AND 3 MONTHS Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

HISTOLOGICAL ABNORMALITIES AT 3 MO Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

PARTIAL FOOT PROCESS EFFACEMENT BY ELECTRON MICROSCOPY AT 3 MO Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

MIR-17~92 LOSS IN NEPHRON PROGENITORS Evidence for glomerular disease: (1) Albuminuria (with heterozygous and homozygous loss of miR-17~92) (2) Focal glomerulosclerosis (3) Partial foot process effacement (4) Decreased renal function

HOW DOES MIR-17~92 REGULATE NEPHRON NUMBER AND PATTERN? N-myc and/or C-myc (ChIP) Candidate downstream targets: Bim, PTEN, p21 RNA sequencing approach to identify novel targets. Regulation of: -apoptosis/cell death -nephron differentiation -renal size Feingold syndrome patients: Renal US Urine protein to creatinine

HIGH THROUGHPUT RNA SEQUENCING OF CONTROL AND MUTANT KIDNEYS April Marrone and Dennis Kostka

VALIDATION OF MOST UPREGULATED TRANSCRIPTS PREDICTED TO BE MIR-17~92 TARGETS

CANDIDATE MIR-17~92 TARGETS Olive et al (2010). Int J Bioch & Cell Biol, 42(8): X

INCREASED P21 EXPRESSION IN MUTANTS Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi /ASN

MIR- 17~92 IN KIDNEY DEVELOPMENT AND DISEASE miR-17~9 2 is required in nephron progenitors for normal renal development: Nephron number and pattern Ablation of miR-17~92 in nephron progenitors and their derivatives results in kidney disease: Renal disease and renal hypodysplasia Outstanding questions: Which miR (or miRs) in the cluster are responsible for this phenotype? Is there an intrinsic defect in nephron progenitors? Eg. specification or self-renewal? What are the downstream targets of miR-17~92 in the kidney?

ACKNOWLEDGMENTS University of Pittsburgh/CHP Yu Leng Phua April Marrone Jessica Chu Andrew Bodnar Collaborators: Carl Bates Sunder Sims-Lucas Donna Stolz Sheldon Bastacky Dennis Kostka Collaborators: Cliff Tabin Andrew McMahon Funding: NIDDK- R00DK Pittsburgh Center for Kidney Research Pilot Project – NIDDK P30 DK March of Dimes Basil O’Connor Starter Scholar Research Grant Norman S. Coplon Extramural Grant

Thanks